Gibbon Necropsy Protocol

Snow Leopard SSP Gross Necropsy Protocol Updated: September 2017

A necropsy examination is one of our most important, occasionally our only, and often our last opportunity to collect valuable diagnostic and archival samples after an animal dies. Listed below is a sample necropsy report and list of recommended tissues for routine collection during a necropsy examination.

1) Collect two sets of tissues for formalin fixation 2) Submit one set to your referral pathologist 3) Permanently archive one set at your institution. 4) Send copies of your completed gross and histologic necropsy reports (using your standard forms or the attached form with inclusion of results from ancillary diagnostics) to the SSP Snow Leopard Veterinary Advisor and Pathology Advisor listed below. 5) Request that your referral pathologist/pathology lab permanently archive the paraffin blocks and glass slides from these cases. If they are unable to perform this service, please contact the SSP Pathology Advisor to discuss options (DO NOT forward formalin fixed tissues).

It may not always be possible to collect all of the recommended samples from each animal. However, the more consistently these goals can be achieved and reports generated the greater the chance that we will accurately identify diseases and disease trends in our collections. Also, whenever possible, submission of a complete set of tissues for histologic processing and review is recommended to provide the best data from which to draw conclusions and add to baseline information.

The submitted information will be used to monitor and manage captive collections of Snow Leopards in the United States.

SSP Coordinator: SSP Pathology Advisor: Jay Tetzloff D. McAloose, VMD, Dipl ACVP Miller Park Zoo Wildlife Conservation Society 1020 S. Morris Avenue Zoological Health Program, Pathology Bloomington, IL 61701 2300 Southern Blvd Phone: 309-434-2825 Bronx, NY 10464 Fax: 309-434-2823 ph: 718-220-7105 [email protected] fax: 718-220-7126 [email protected]

SSP Veterinary Advisor(s): Tim Georoff, VMD, Dipl. ACZM Nancy A. Carpenter, DVM, MS Kelly Helmick, DVM, MS, Dipl. ACZM Associate Veterinarian Director of Animal Health Supervisory Veterinary Medical Officer Philadelphia Zoo Utah’s Hogle Zoo Smithsonian Conservation Biol Institute 3400 W. Girard Ave. 2600 E. Sunnyside Ave. 1500 Remount Rd Philadelphia, PA 19104-1196 Salt Lake City, UT 84108 MRC 5533 ph: 215-243-5302 ph: 801-584-1795 Office Front Royal, VA 22630 fax: 215-243-021 fax: 801-584-1793 fax ph: 540-635-0102 [email protected] [email protected] [email protected]

Special tissue request: **There are currently no special requests. Please contact the Snow Leopard SSP Coordinator or Veterinary Advisor with any special or research requests.

General Necropsy Information : 1) Tissue collection 1) Collect normal and abnormal tissue samples from all major organ systems 2) Tissue samples should be no thicker than 0.5 cm 3) Tissue: 10% neutral buffered formalin (ratio) = 1:10 3.i) Multiple tissue samples can be placed in a single bucket, but maintain (1:10 tissue to formalin) ratio 4) Permanently archive one set of tissues. Tissues can be saved or shipped in smaller volumes of formalin once adequately fixed 2) Specific tissue collection and fixation recommendations 1) Lymph nodes: Label as to location (e.g. mesenteric, mandibular) when grossly abnormal or in cases of suspect hematopoietic or lymphoid disease 2) Heart: Collect 3 longitudinal sections (minimum) such that atrium, ventricle and valve from right and left freewalls (include papillary muscle) and interventricular septum (include right AV valve and aortic outflow tract) are collected 3) Gastrointestinal tract: For oral cavity, document (photograph, include ruler) and measure any papillomas (generally found on ventral surface of tongue). To sample papillomas for histology, try to include the entire lesion superficially and to a depth of at least 5 mm. Also take samples of the papilloma for freezing (ultralow temp). Open the entire tract along its long axis. Collect 2-3 sections (3- 4 cm long) from each part of tract. Label as to location when grossly abnormal or in cases of suspect gastrointestinal tract disease. Note content. 4) Urogenital system: 4.i) Testes: Measure and record length, width, thickness of both testes. Cut tunica and then cut each testis longitudinally to ensure adequate tissue fixation 4.ii) Ovaries: Testes: Measure and record length, width, thickness of both ovaries. Cut each ovary longitudinally to ensure adequate tissue fixation 4.iii) Urinary tract (4.iii.1) Collect urine for urinalysis and cytology (4.iii.2) Collect any crystals or calculi for mineral composition analysis 5) Endocrine organs: Collect organs from both the left and right sides. Longitudinally section adrenal glands to assess cortical and medullary tissue. 6) Brain: Fix whole if possible. Place in separate bucket to improve rapidity of initial fixation if possible. The brain should then be reunited with the rest of the tissues when archived. 6.i) If only half brain can be collected, section brain longitudinally along midline 7) Eye: Fix whole (not punctured and do not inject with formalin) 8) Neonates and fetuses: 8.i) Measure crown-rump length 8.ii) Verify sex 8.iii) Collect umbilical stump and surrounding skin and subcutis 8.iv) Evaluate for malformations (e.g. cleft palate, cardiovascular abnormalities, coloboma) 8.v) Evaluate lungs (e.g. float in formalin if breathing occurred; sink if not inflated (e.g. stillborn), diseased (e.g. pnemonia)) 8.vi) Evaluate for evidence of nursing (presence of milk fluid and/or curd in stomach and presence of “milk stool” (yellow-white semisolid material in colon) with absence of meconium (greenish/brown pasty material) 8.vii) Collect fetal membranes if available and take multiple 0.5 cm samples from various sites 8.viii) Assess hydration status 3) Diagnostic samples – general tissue handling for surveillance/monitoring or to test lesions Note: Submit samples (e.g. culturettes, fresh tissue, feces) on dry ice or ice packs to internal or external labs as necessary for ancillary diagnostic testing; package and ship per your standard procedures and in compliance with local/regional/national shipping requirements 1) Infectious disease or molecular diagnostics (e.g PCR, DNA sequencing) 1.i) Store at –70°C (to maintain DNA, proteins, RNA) 1.ii) Collect samples per research request or based on presence of lesions 2) Toxicology 2.i) Store tissues or samples at –20°C 2.ii) In cases of suspected toxicity, contact a toxicologist for recommendations relative to tissue collection, preservation, test type and shipping instructions 2.iii) For generic toxicologic sample collection, collect two sets (1 in aluminum foil, 1 in plastic or glass) 5 grams each, of the following tissues: (2.iii.1) Stomach contents (2.iii.2) Adipose tissue (2.iii.3) Kidney (2.iii.4) Liver (2.iii.5) Eye (2.iii.5.a) if possible, collect, store, test aqueous and vitreous samples separately (contact lab for recommendations); if not possible, collect, store, test entire eye 4) Frozen Tissues – General Archiving 1.i) Collect, freeze: liver, spleen, brain (minimum tissue set; minimum one piece each recommended) 1.ii) Collect, freeze lesions (in addition to a sample/s submitted for diagnostic testing) (1.ii.1) Store at –70°C or in liquid nitrogen in appropriate 1.5 ml cryovials or small freezer- appropirate bags (1.ii.1.a) Store at conventional freezer temperatures if ultralow freezer space not available. iii. Note: the useable lifespan of these tissues is protracted due to the freeze/thaw cycle of the storage method. Gross Necropsy Report – General information – (ok to provide specimen report) Species: Sex (M/F/U): Common Name: Date of birth/age: Institution/Owner: Date of death: Accession/ID No: Type of death (Euth/Natural): Studbook No.: Prosector: Other ID No: Necropsy Date: House Name: Necropsy Location: Enclosure ID/indoor/outdoor: Body weight: Captive Born or Wild Caught: Pathologist: Parent or hand-reared: Pathologist Phone Number: Dam: Sire: Environmental/weather conditions at time of death: Movements or relocations (date, from and to): Diet: (electronic copy ok to include)

Contraceptive history (contraception: Y/N; type; date):

Proven breeder (Y/N; number of offspring produced; birth history (live, stillborn, abortion, etc.)

Clinical history and past treatments (add additional sheets for clinical history or ancillary diagnostics (e.g. serum chemistries, radiography) as necessary): (Provide medical history report (PDF) or similar) Tissue examination and collection checklist -20 -20 TISSUE GA FF Histo Photo TISSUE GA FF Histo Photo -70 -70 GENERAL-external ABDOMEN Oral cavity & teeth Diaphragm Tonsils Stomach Skin Small intestines Nails Large intestines Subcutis Pancreas Skeletal muscle Spleen Peripheral nerves Liver & gall bladder Mammary gland Lymph nodes Umbilicus Aorta & vessels BONES & JOINTS Kidneys Bone marrow (femur) Ureters Bones Urinary bladder Hips Urethra Knees Adrenal glands Tarsi Ovaries Shoulders Oviduct/Uterus Carpi Vagina/vulva Atlantooccipital Testes CAVITIES Access sex gland Thoracic cavity Penis/prepuce Abdominal cavity HEAD PLUCK Eyes Tongue Ears & bullae Thyroids/parathyroids Skull/nasal cavity Esophagus Brain/Meninges Trachea & Lungs Pituitary gland Heart/Pericardial sac SPINE Aorta & other vessels Vertebral column Thymus/lymph nodes Spinal cord GA = Gross appearance: NGL=no gross lesions; AB=abnormal; NE=not examined; NF=not found; NP=not present FF = Tissue fixed in formalin: + = tissue collected and fixed Histo = Tissue submitted for histology: + = tissues submitted AD = Ancillary diagnostics: + = ancillary diagnostic performed (please include results w/report) PHOTO = Photograph: + = PHOTO taken -20°C /-70°C = Frozen tissue temperature: please list storage temp as –20°C, -70°C or other temp if applicable

Gross Descriptions: Please describe all abnormal gross findings, including dimensions (cm x cm x cm). General condition (include assessment of body and post-mortem condition, skin, SQ fat stores, body orifices):

Musculoskeletal system (axial, appendicular, tympanic bullae, spinal column, joints, muscle):

Body cavities (thoracic, abdominal, pericardial, also assess fat stores in these locations):

Hemolymphatic systems (tonsil, lymph nodes, spleen, thymus, bone marrow):

Respiratory system (nasal passages, pharynx, larynx, trachea, bronchi, lungs):

Cardiovascular system (heart, valves, chambers, pericardial sac, vessels):

Digestive system (tongue, oral cavity, teeth, esophagus, stomach, small and large intestines, anus, liver, gall bladder, pancreas):

Urinary system (kidneys, ureters, urinary bladder, urethra):

Reproductive system (testes/ovaries, uterus, cervix, penis/vagina, accessory sex organs, mammary glands, placenta, fetus):

Endocrine system (adrenal glands, thyroids, parathyroids, pituitary gland):

Nervous (brain, spinal cord, peripheral nerves) and special senses (eyes, ears):

Gross Diagnoses (please add additional diagnoses if necessary):

Gross Comments:

Results of ancillary post-mortem diagnostics (e.g. cytology, fluid analysis, urinalysis, bacteriology, toxicology, virology, parasitology):

Note: Please also attach/forward gross and histology report to veterinary and pathology advisors when available.